The long-term objective of this research is to understand the function of scavenger receptor class B type I (SR-BI) in the delivery of cholesteryl ester (CE) from high density lipoproteins (HDL) to the plasma membrane for its subsequent metabolism. SR-BI is the HDL receptor that regulates HDL cholesterol metabolism and is directly linked to the ability of HDL to be athero-protective. Therefore, understanding how SR-BI delivers HDL-CE to a site in the plasma membrane that allows for its efficient metabolism is key to developing methods for prevention of cardiovascular disease. This proposal consists of three primary objectives that will evaluate the mechanisms of SR-BI-mediated uptake and hydrolysis of HDL-CE. Aim 1 will investigate the structural organization of SR-BI at the plasma membrane. Goal 1 will use fluorescence resonance energy transfer techniques to extend our understanding of the oligomeric organization of SR-BI in intact cells in the presence and absence of ligands. Additionally, mutagenesis studies will help delineate the region within SR-BI that is required for its oligomeric properties. Goal 2 will examine the functional domains in the extracellular domain of SR-BI using tryptophan quenching by spin labeled fatty acids. Aim 2 is designed to test the hypothesis that SR-BI modifies the composition of membrane phospholipids to favour selective uptake of HDL-CE. Goal 1will compare liver/adrenal membrane phospholipid profiles in wild-type and SR-BI knock-out mice using tandem mass spectrometry. Then, we will exploit adenovirus-mediated liver/adrenal expression of SR-BI and its mutants to investigate Ijhe structural regions/functions of SR-BI that are required for changes in phospholipid speciation. Goal 2 will examine the effects of SR-BI-independent alterations in membrane phospholipids on selective uptake efficiency, either by transfection of cells with desaturases/elongases or by reconstitution of SR-BI into liposomes with different ratios of sphingomyelin: cholesterol. Aim 3 will help us understand the mechanisms of HDL-CE delivery and hydrolysis at the plasma membrane. We hypothesize that an accumulation of CE in the plasma membrane will prevent further uptake of HDL-CE. Therefore, in Goal 1, vesicle reconstitution, in addition to the use of a novel fluorescent lipid, will let us measure the accumulation of CE at the plasma membrane in the presence of wild-type or a non-functional SR-BI mutant. Goal 2 will examine the co-localization of hormone-sensitive lipase with SR-BI at the plasma membrane for hydrolysis of HDL-CE in adrenal cells and tissues: Together, these studies will provide new mechanistic information about the role of SR-BI in HDL-CE selective uptake and hydrolysis, and shed new insights into cholesterol metabolism and protection against atherosclerosis.